Xtt assay

Without PBMC co-culture, NPC cells were seeded in a 96-well plate at a density of 1000 cells/well/100 μL RPMI-1640 complete medium. The cell proliferation was evaluated by using the XTT assay (Roche), as per manufacturer’s instructions, for a consecutive 5 days. With PBMC co-culture, PBMCs in the supernatant were removed, and the tumor cells were washed with PBS twice to remove the remaining PBMCs and dying cells. Fresh RPMI-1640 complete medium was added to each well with adherent tumor cells. The read absorbance (492 nm as the absorbance wavelength and 620 nm as the reference wavelength) was evaluated by using the XTT assay (Roche), as per manufacturer’s instructions, at 24 hours and 48 hours post-PBMC co-culture. The relative absorbance was calculated by subtracting the read absorbance from the absorbance of an empty well. Relative cell survival was normalized by the relative absorbance of viable cells without PBMC co-culture.

Loss‐of‐function experiments made use of PHGDH si‐RNA (catalogue nos. SASI_Hs01_00041882 and SASI_Hs01_00041884; Sigma) and negative‐control si‐RNA (D‐001810‐10; Thermo Fisher Scientific, Waltham, MA, USA). PHGDH inhibitors included CBR‐5884 (Focus Biomolecules, Plymouth Meeting, PA, USA) and NCT‐503 (AOBIOUS, Gloucester, MA, USA) as previously described (Pacold et al., 2016). DMSO was used to dilute both inhibitors following the manufacturer's recommendations. Cell proliferation was determined with XTT assays (Roche Applied Science, Tokyo, Japan) according to the manufacturer's instructions. Cell apoptosis was measured by flow cytometric determination using the CytoFLEX analyzer (Beckman Coulter, Brea, CA, USA) and a FITC Annexin V Apoptosis Detection Kit (BD Biosciences, Bedford, MA, USA) as per the manufacturer's recommendations. The positive control utilized 5 μg·mL⁻¹ cycloheximide (Sigma). Colony formation assays were previously described (Yoshino et al., 2017b).